Reactivation of latent cytomegalovirus (CMV) occurs frequently in immunosuppressed recipients of allogeneic hematopoietic stem cell transplant (HCT) as a consequence of deficient T cell immunity, and is responsible for substantial morbidity and mortality. Antiviral drug therapy with ganciclovir has reduced the incidence of early CMV disease in transplant recipients but causes myelosuppression and only transiently suppresses CMV replication. A strategy that restores T cell immunity could provide a durable alternative therapy to toxic antiviral drugs for immunocompromised HCT recipients at risk for CMV disease. Studies supported by this project have investigated the immunobiology of CMV infection and developed strategies for specific adoptive immunotherapy with CD8+ T cell clones to correct deficiencies of T cell immunity that permit the progression of CMV infection. Clinical trials of this approach have demonstrated that adoptive T cell therapy for CMV is safe and can restore functional T cell immunity in a subset of patients. The major obstacles to effective immunotherapy of CMV have also been identified and include poor persistence and function of transferred T cells due to intrinsic properties of cultured T cells that interfere with their capacity to survive in vivo and the effects of corticosteroids administered to patients to treat GVHD. Studies in the previous funding cycle have defined properties of CD8+ T cells that enable the selection of those with the intrinsic capacity to persist and revert to the T cell memory pool in vivo, and identified a strategy to selectively knock out glucocorticoid receptor function in CMV-specific T cells, rendering the cells resistant to suppressive effects of corticosteroids. This proposal will build on these efforts to develop T cell immunotherapy for controlling persistent CMV replication. The specific aims are: 1). To evaluate in vitro the function and phenotype of macaque and human CD8+ CMV-specific T cells engineered to lack glucocorticoid signaling after genome editing of the glucocorticoid receptor. 2). To evaluate the safety and in vivo function of adoptively transferred CD8+ CMV-specific T cell clones with an edited glucocorticoid receptor gene in non-human primates. 3). To determine whether adoptively transferred CMV-specific T cells with an edited glucocorticoid receptor gene respond to and prevent progression of CMV infection in non-human primates that are immunosuppressed with prednisone. PUBLIC HEALTH RELEVANCE: Cytomegalovirus infection is a major cause of morbidity and mortality in patients who undergo allogeneic hematopoietic stem cell transplantation and require systemic corticosteroids to treat graft versus host disease. Studies supported by this project have investigated the immunobiology of CMV infection in immunodeficient transplant recipients, and developed adoptive immunotherapy with CMV-specific T cells to restore protective immunity. The proposed studies will investigate the potential for adoptively transferred CMV- specific CD8+ T cells that have been selected for the ability to establish long-lived memory cells and edited at the glucocorticoid receptor gene using zinc finger nucleases to establish durable T cell immunity that is resistant to the immunosuppressive effects of glucocorticoids.